The invention relates to tandem mass spectrometers with a spatially separated mass selector and mass analyzer, with a cell for fragmentation by electron transfer dissociation (ETD). Investigation of the structures and functional activities of proteins, and also of other biopolymers, is largely based on so-called tandem mass spectrometry, which not only provides spectra of the protein ion mixtures but also allows individual protein ions to be selected as “parent ions” for fragmentation, isolated from other ions and then fragmented so that the fragment ions produced can be measured in a mass spectrum. Depending on the type of fragmentation, these fragment ion mass spectra especially provide information on the primary and secondary structures of the proteins that allows not only the genetically determined basic structure of their amino acids (the “sequence”) to be identified according to type and localization, but also other modifications that are important because they change the function of the protein (“posttranslational modifications”, PTM).
The individual steps of tandem mass spectrometry are first selection and isolation of the parent ions, then fragmentation, and finally mass analysis of the fragment ions. In storage mass spectrometers such as ion traps, these steps can be carried out consecutively in the same storage unit (“tandem-in-time”). But the selection and isolation of the analyte ions to be fragmented can also be carried out in a first mass analyzer, the “mass selector”, with fragmentation in a special cell which may or may not be identical with the mass selector, and with mass analysis in a second spatially separated mass analyzer (“tandem-in-space”). The invention relates to such a tandem mass spectrometer with spatially separated mass selector and mass analyzer.
The use of tandem mass spectrometry in temporal sequence in storage mass spectrometers is extraordinarily widespread and has been shown to be very useful up to a certain point. This applies both to tandem mass spectrometry in ion cyclotron resonance mass spectrometers (ICR-MS), where ultra-high mass resolutions can be achieved, and also to the use of RF ion trap mass spectrometers (IT-MS) with limited mass resolution and mass accuracy. Both types of tandem mass spectrometer have limitations, however, which concern partly the measuring speed (ICR-MS is slow), partly the mass range (IT-MS has a lower mass cut-off) and partly the mass accuracy (IT-MS has limited accuracy), and are largely determined by the type of fragmentation used. Nowadays, tandem mass spectrometry is often coupled with relatively fast separation methods for the substances, for example nano-liquid chromatography (nano-HPLC) or capillary electrophoresis (CE), so the substances of the short substance peaks can only be analyzed for a few seconds at most in the mass spectrometer. Measuring speed thus plays an important role; moreover, it is becoming increasingly important to achieve, in particular, high mass accuracy and to acquire a fragment ion spectrum that is as complete as possible, including the light fragment ions.
Because of the high demands placed on mass accuracy and speed in the measurement of fragment ions, it has proved advantageous to carry out the fragmentation in a separate cell and to then measure the fragment ions produced in an especially suitable mass analyzer, particularly a time-of-flight mass spectrometer with orthogonal injection of the ions (OTOF-MS). Modern Kingdon ion traps or ion cyclotron resonance mass spectrometers can also be used as mass analyzers owing to their high mass resolution, but only if the measuring speed is of secondary importance.
The success of tandem mass spectrometry depends on the fragmentation methods used. There are essentially only two fundamentally different types of fragmentation available for proteins or similar biopolymers: “ergodic” and “electron-induced” fragmentation. For each of these there are many different favorable embodiments. The various individual methods often have deficiencies with respect to the mass range of the fragment ion spectra, the fragmentation speed and, in particular, the quality of the fragment ion spectra, and therefore how well they can be evaluated. The quality of the fragment ion spectra may be defined by a high yield of terminal fragment ions, producing ladders covering as equally as possible all amino acids, low chemical background noise, and low amounts of internal fragments which greatly disturb the evaluation.
These fragmentation methods will therefore be discussed here before the invention can be explained. The reaction cells for the fragmentation always have the form and function of RF ion traps; the fragmentation of the selected parent ions takes place inside these reaction cells.
As has been noted above, two fundamentally different kinds of fragmentation are now available in reaction cells of various kinds: “ergodic” fragmentation and “electron-induced” fragmentation. These lead to two significantly different kinds of fragment ion spectra, whose information content is complementary and which produce particularly detailed information on the structures of the analyte ions when both types of fragment ion spectra are measured.
The term “ergodic” fragmentation of analyte ions (sometimes also called “thermal”) here means a fragmentation where a sufficiently large excess of internal energy in the analyte ions leads to fragmentation decay via a “metastable state” with a decomposition half-life of between several and several hundred microseconds (or more). The excess energy can, for example, be produced by a large number of inelastic collisions of the analyte ions with a collision gas; or by the absorption of many photons from an infrared radiation source.
The conventional type of fragmentation of the analyte ions in RF ion traps is ergodic fragmentation by collisions of the somehow accelerated analyte ions with the collision gas contained in the ion trap. In this process, the excess internal energy of the moving analyte ions is collected by collisions with the stationary collision gas molecules. In order for the collisions to be able to pump any energy into the analyte ion at all, they have to occur with a minimum of collision energy. Since gentle collisions of the analyte ions with the collision gas can always cause an internal cooling by removing energy, there is always competition between “heating” and “cooling”; physically heavy ions, in particular, require a higher collision energy for the heating than light ions. At a specified density of the collision gas and specified kinetic energy of the collisions, for physically heavy analyte ions above a certain mass it is always the cooling which predominates; these analyte ions cannot be fragmented at all in this way.
In three-dimensional RF ion traps (“3D ion traps”) consisting of a ring electrode and two end cap electrodes, the collision energy is generated in a conventional way by limited resonant excitation of the secular ion oscillations of the parent ions with a dipolar alternating voltage on the end cap electrodes. This leads to many collisions with the collision gas without removing the ions from the ion trap. The parent ions can accumulate energy in the collisions, which finally leads to ergodic decomposition of the parent ions and the creation of fragment ions. The fragment ions are often also called “daughter ions”. Until a few years ago, this collision-induced dissociation (CID) was the only known type of fragmentation in ion traps.
This collision-induced dissociation in three-dimensional RF ion traps also has disadvantages, however. For physically heavy analyte ions, it is necessary to set the RF voltage for storing the ions at a very high level in order to produce sufficiently hard collision conditions. This results in a very high minimum mass threshold for the ion trap. Ions with masses below this mass threshold can no longer be stored; they are lost. The fragment ion spectrum therefore only starts at a mass which, according to a conventional rule of thumb, is about one third of the charge-related mass m/z of the analyte ion; the fragment ion spectrum can no longer provide any information on the light fragment ions because these ions are lost. Multiply charged, physically heavy analyte ions with physical masses of many thousand daltons regularly have a relatively low charge-related mass m/z of about 700 to 1200 daltons, owing to the large number of protons; these analyte ions cannot be fragmented at all because the RF voltage cannot be set high enough to produce sufficient numbers of high-energy collisions.
There thus remains the big disadvantage of conventional collision-induced dissociation that with physically heavy analyte molecules above about m=3000 daltons, the corresponding analyte ions can hardly be fragmented at all with the conventional collision method.
An ergodic fragmentation which does not have this disadvantage has been described in the document WO 02/101 787 A1 (S. A. Hofstadler, and J. J. Drader). This method applies infrared multi-photon dissociation (IRMPD), which is known from ICR mass spectrometry, to RF ion traps as well. The infrared radiation here is introduced into a three-dimensional RF ion trap via an evacuated hollow fiber with an optically reflective internal coating, through the ring electrode which is perforated for this purpose. This type of fragmentation is advantageous because it can be carried out at low RF voltages; the small fragment ions are then also stored. The internal surfaces of the ion trap must be kept extremely clean because any molecules adhering to the walls are detached by the irradiation of the infrared photons and then react with the stored analyte ions in a variety of ways. This is the main reason why there are still no commercially available ion trap mass spectrometers with this type of fragmentation.
Another ergodic fragmentation method in ion traps has recently been proposed which also does not have the disadvantage of being unable to fragment heavy analyte ions or store light fragment ions. In this case, stationary stored analyte ions are bombarded with preferably anti-polar, if possible mono-atomic, collision ions with adjustable kinetic energy. Since the RF voltage here can be set very low, heavy analyte ions can be successfully fragmented due to the higher energy transfer per collision, and on the other hand, very light fragment ions can be trapped and measured.
This means there is now at least one method of ergodic fragmentation available which no longer has any significant disadvantages.
Now we turn to the electron-induced fragmentation methods. About ten years ago, a completely new type of fragmentation of protein ions was discovered: a non-ergodic fragmentation induced by the capture of low-energy electrons (ECD=“electron capture dissociation”). By the direct neutralization of an associated proton at one position in the amino acid chain, which then gets lost as a radical hydrogen atom, the potential equilibrium of the protein ion in the vicinity of the neutralized proton is disturbed so much that a cleavage of the amino acid chain is induced by corresponding rearrangements. The cleavage does not affect a peptide bond, but predominantly an adjacent bond, leading to so-called c and z fragment ions. In contrast with ergodic fragmentation, this fragmentation occurs spontaneously, in less than 10−8 seconds.
This type of fragmentation is particularly easily to carry out in ICR mass spectrometers because the low-energy electrons from a thermionic cathode can easily be supplied along the lines of magnetic force to the stored cloud of analyte ions. But ECD fragmentation can only be used with some difficulty in RF ion traps because the strong RF fields do not easily allow the low-energy electrons to come very close to the cloud of analyte ions. Nevertheless, there are a number of solutions for ECD fragmentation in RF ion traps, but they each require costly apparatus and have not yet achieved a satisfactory sensitivity.
A few years ago, a method for the fragmentation of ions in RF ion traps was presented which produces fragmentations similar to electron capture dissociation (ECD) but by different reactions: “electron transfer dissociation” (ETD). ETD can easily be carried out in ion traps by adding suitable negative ions to the stored analyte ions. Methods of this type have been described in U.S. Pat. No. 7,456,397 B2 (R. Hartmer and A. Brekenfeld) and US 2005/0199804 A1 (D. F. Hunt et al.). As in ECD, the fragment ions here belong to the so-called c and z series, and are therefore very different to the fragment ions of the b and y series obtained by ergodic fragmentation. In particular, all the side chains, which are always lost with ergodic fragmentation, are preserved during electron transfer dissociation, including the important posttranslational modifications such as phosphorylations, sulfurylations and glycosylations.
The fragmentation of protein ions by electron transfer (ETD) in an RF ion trap is brought about in a very simple way by reactions between multiply charged positive protein ions and suitable negative ions. Suitable negative ions are usually aromatic radical anions, such as those of fluoranthene, fluorenone, anthracene or other polyaromatic compounds; but there are also some non-aromatic radical ions, like e.g. those of cyclooctatetradiene, which can be used successfully for ETD. In radical anions, the chemical valences are not saturated, so they can easily donate electrons and thus achieve an energetically favorable non-radical form. They are generated in NCI ion sources (NCI=“negative chemical ionization”), most probably by electron capture or by electron transfer. NCI ion sources are constructed, in principle, like ion sources for chemical ionization (CI ion sources), but operated differently in order to obtain large quantities of low-energy electrons. NCI ion sources are also called electron attachment ion sources.
Ergodic fragmentation initially cleaves all posttranslational modifications that are only weakly bonded, such as phosphorylations, sulfurylations and glycosylations, and essentially displays the naked sequences of the unmodified amino acids of the analyte ions. Therefore, neither the existence, nor the type or position of the posttranslational modifications can be identified. In contrast with ergodic fragmentation, these modifications are not cleaved off by electron-induced fragmentation. In comparison with the ergodically obtained fragment ion spectra, an additional mass at an amino acid thus shows both the type and also the position of the modification. These extraordinarily important investigative results can only be obtained in this favorable and simple way by comparing both types of fragment ion spectra.
Particularly for investigating posttranslational modifications (PTM), it is therefore necessary nowadays to acquire ion spectra both by ergodic fragmentation and by electron-induced dissociation in parallel. Both types of fragment ion spectra should also satisfy the highest quality requirements. A modern tandem mass spectrometer for bio-analyses must therefore offer both types of fragmentation in methods that are as free from deficiencies as possible. But also for other analyses, such as de novo sequencing, a comparison of good quality fragment ion spectra obtained using both ergodic and electron-induced methods is advantageous or even absolutely imperative.
Until now, only 2D ion traps have been used as separate ETD fragmentation cells in tandem mass spectrometers with high-resolution mass analyzers. Although ETD fragmentation can also be performed in 3D ion traps, the commercial instruments so used are limited to those mass spectrometers which simultaneously use this 3D ion trap as the sole mass analyzer for measuring the fragment ion spectra. Their design does not allow the fragment ions to be transferred into a different mass analyzer.
The ETD fragment ion spectra from 2D ion traps are not of a very high quality; they are enriched with high proportions of unused analyte ions and fragment ions of the second and third generation (with many so-called “internal fragment ions”, which are not terminal) and are also not very sensitive.
Three-dimensional ion traps have occasionally been coupled to mass analyzers with a higher mass resolution, but they have never been used for an ETD fragmentation. Such use of a 3D ion trap for an (albeit only ergodic) fragmentation relates to a commercially available Shimadzu tandem mass spectrometer, in which the 3D ion trap is used to axially inject the ions from its interior into a time-of-flight mass spectrometer by means of a high-voltage pulse. Ergodic fragmentation is possible in this 3D ion trap; a version with electron-induced fragmentation is neither known nor available. This mass spectrometer requires separate filling of the 3D ion trap for each time-of-flight mass spectrum. It is a relatively slow and insensitive instrument because, in each individual time-of-flight mass spectrum, the dynamic range of measurement is limited by the small conversion width of current transient recorders, and the 3D ion trap can therefore only be operated with a limited number of analyte ions.